Project Summary Project Title: High-stability solid-state cooled Cryo-TEM transfer holder Company Name: Hummingbird Precision Machine Co., dba Hummingbird Scientific Principal Investigator: Daan Hein Alsem Summary: Cryo-TEM or electron cryotomography (ECT) produces high-resolution 3D views of biological samples in hydrated and functional state. This microscopy technique has a leading role in structural characterization efforts of biological samples, including: intact cells, organelles, macromolecular complexes, and proteins, facilitating basic knowledge and drug discovery. Over the last decade, the resolution of cryo-TEM has seen an unprecedented improvement down to below 3 Angstrom because of the advent of direct-electron detection cameras, which are orders of magnitude more sensitive than previous TEM camera technology and new high-throughput loading mechanisms in the latest generation of dedicated, but expensive, Cryo-TEM microscopes. However, the resolution achieved with these new dedicated TEMs is not better than the resolution capability of standard TEMs installed twenty years ago. There are still hundreds of these previous models of TEMs used for imaging of biological specimens. These labs can (and in many cases have) purchase the latest generation sensitive cameras for these microscopes, which makes that these older microscopes are at that point not limited by their electron-optics, or their camera, but by the cryo-TEM sample holder. The cryo- TEM holder technology used today relies on evaporation of LN2 in a geometrically asymmetric Dewar attached to the sample to cool the sample, which causes image instability and vibration, specifically when changing the tilt angle when acquiring tomograms. Because of shifting mass in the LN2 Dewar and the LN2 encountering less cooled surfaces when tilting resulting in excess boiling, the image stability in these TEM holders is far from perfect. This project will provide scientists with a TEM cryo-transfer sample holder that eliminates these image instability and vibration limits and allows the previously installed cryo-TEMs to achieve the performance similar to or matching the latest generation of dedicated and very expensive cryo-TEMs. We are proposing to achieve this by using a cryo-TEM sample transfer holder that has a rotationally symmetric solid-state cooling block to cool the sample. Because of the symmetry there are no changes in forces on the holder when tilting and the solid-state cooling method assures there is no excess boiling when LN2 is moving around during tilting of the specimen. This eliminates sample instability and vibration that arise during tilting of the holder during acquisition of cryo-TEM tomograms. As an added benefit, this innovation also provides a much longer cold life of the sample (>12 hours) than current generation Cryo-TEM holders. We will achieve this by using an ultra-cooled solid-state cold mass that has no boiling or liquid mass but is instead cold- charged to temperatures below liquid nitrogen temperatures (~50K) to provide long cold-life of the TEM. This adds up to a TEM specimen holder that can provide higher image stability and operate longer without having to be cold-recharged. This product is aimed at scientists in the hundreds of cryo-TEM labs where purchasing an expensive dedicated cryo-TEM is not a realistic proposal. These labs will still propel their research with this new side-entry TEM cryo transfer holder as it will give them a better quality and higher resolution cryo-TEM image that is similar to what they would achieve if they would purchase a dedicated TEM.